Waters Protein-Pak SEC Columns User Manual
[ CARE AND USE MANUAL ]
PROTEIN-PAK SEC COLUMNS
I. INTRODUCTION
This Care and Use Manual contains information pertaining to the use of
the following Protein-Pak™ SEC Columns:
Protein-Pak 60 7.8 mm (i.d.) x 30 cm, Steel (P/N
WAT085250)
Recommended for use with biopolymers such as peptides, polypeptides,
steroids and small proteins. T he pore size distribution provides
excellent separation in the following molecular weight ranges: Native
Globular 1000-20,000 and Random Coil 600 - 8000. Shipped in
100 % m ethanol .
Protein-Pak 125 7.8 mm (i.d.) x 30 cm, Steel (P/N
WAT 084601)
Especially suited for separation of biopolymers such as proteins and
enzymes in the following molecular weight ranges: Native Globular
2000 - 80,000 and Random Coil 1000 - 30,000. Shipped in
100 % m ethanol .
Protein-Pak 300 SW 7.8 mm (i.d.) x 30 cm, Steel (P/N
WAT080013)
Protein-Pak 200 SW 8.0 mm (i.d.) x 30 cm, Glass (P/N
WAT 01178 6)
Protein-Pak 300 SW 8.0 mm (i.d.) x 30 cm, Glass (P/N
WAT011787)
CONTENTS
I. INTRODUCTION
II. PREPARATION FOR OPERATION
a. Steel Column Installation
b. Glass Column Installation
c. Solvent Requirements
d. Buffers
e. Protein Denaturants
f. Equilibration
g. Column Purge Volumes
III. CARE AND USE
a. Sample Preparation and Filtration
b. Precautions
c. Storage Consideratons
IV. TROUBLESHOOTING
a. Service and Applications Information
b. Column Efficency
c. Test Conditions
The columns of choice for use with larger biological active molecules
in the molecular weight range of Native Globular 10,000 - 400,000
and Random Coil 2000 - 150,000. Shipped in 0.05% sodium azide in
water solution.
Protein-Pak SEC Columns 1
[ CARE AND USE MANUAL ]
maximum column eciency
Critical Distance to be determined by each
II. PREPARATION FOR OPERATION
a. Steel Column Installation
Remove the end plugs from your colum with a 5/18” wrench and
save them for storage when the column is removed from the system.
The column outlet is indicated by an arrow on the label (showing the
direction solvent should flow). Tighten the fittings 1/4-to-1/2 turn.
DO NOT OVERTIGHTEN – THIS WILL DAMAGE THE FITTING SEAT.
A properly prepared and assembled compression fitting in good
conditions is all that is required.
Follow the next four steps of this procedure if tubing cutting is required
to connect a new steel column or to improve the end connections on your
existing fittings.
1. Using a file with a cutting edg, escribe the circumference of the tubing at
the desired break.
2. Grasp the tubing on both sides of the scribe mark with cloth covered pliers
(to prevent marring the tube surface), and gently work the tube back and
forth until it separates.
3. File the ends smooth and assemble as shown.
Note: Proper positioning of the fetrrule in the fitting seat will prevent
formation of unwanted dead volume which could result in sample mixing.
4. Slide the compression fitting, followed by the ferrule (large end of the
taper first) over the tube. Be certain to bottom the tube in the fitting seat
for which its use is intended to assure a leak-free connection.
Note: Attach a union in place of the column and flush the lines free of
microparticulates before attaching the column.
Figure 1: Ferrule and Compression Screw Assembly
Compression screw or nut
Tube
application (union, column tting, etc.)
End must be straight
and smooth to achieve
Ferrule
b. Glass Column Installation
Waters Protein-Pak 200 or 300 SW glass columns can be connected to
any HPLC or medium-pressure system remembering not to exceed 300 psi
backpressure with the installed glass SEC columns. T he following parts can
be used to adapt these columns to any of our standard Waters systems:
WAZ TO 1/4 UNF Adapter WAT089472
Compression Screw 1/8 o.d. 1/4 - 28 WAT08 8467
Reverse Ferrule 1/8 o.d. WAT088466
Tube Cut 0.125 x 0.009 x 120 WAT 088431
c. Solvent Requirements
Protein-Pak 60 and 125 columns are shipped containing an organic
solvent compatible with water-miscible solvents (such as methanol,
acetonitrile, etc.) The Protein-Pak 200 and 300 SW columns are shipped
in water containing 0.05% sodium azide, added as a bacteriostat. If
an immiscible solvent is to be used for analysis, make the changeover
gradually using at least one itermediate solvent (similar to changing over
solvents during analysis). When changing the organic concentration in
the mobile phase to reduce the flow rate. When adding organic solvents
to aqueous buffer solutions avoid salt precipitation.
d. Buffers
Protein-Pak columns are stable in normal salt buffer solutions such as
sodium sulfate, ammonium acetate, ammonium formate, phosphate
buffers, tris acetate and acetate buffers. Ionic strength equivalent to 0.1 M
minimizes the ionic interaction between the protein sample and the silica
stationary phase. Generally the salt concentrations in aqueous solutions
should be maintained below 0.5 M.
e. Protein Denaturants
Aqueous solutions of SDS, guanidine hydrochloride and urea are
compatible with the Protein-Pak columns although there is a tendency
to display shorter column life when compared to non-detergent
aqeuous solutions. We recommend dedicating columns used with these
solutions rather than changing back to other aqueous solutions with the
same column.
Protein-Pak SEC Columns 2
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